Colorectal cancer (CRC) is a leading cause of cancer related death in the United States. It develops through a multistep process driven by the accumulation of genetic and epigenetic alterations in colonic epithelial cells leading to transformation into cancer cells. The role of epigenetic alterations in CRC is incompletely understood. Hundreds to thousands of genes are aberrantly methylated in CRC, yet the biological significance of most of these events is unknown. The genes that can be inactivated by aberrant DNA methylation include genes in signaling pathways, and those that regulate proliferation and apoptosis, such as the RET conditional tumor suppressor among many others. The biological functions of the genes that are affected by epigenetic alterations suggest that they may be useful for predicting the biological and clinical tumor behavior. Considerable heterogeneity exists among the clinical behavior of CRCs, highlighting the need for more accurate markers of survival and disease recurrence. Studies to date suggest that both genetic and epigenetic alterations can predict response to therapy and clinical outcome independently of clinicopathologic stage, currently the best predictive factor. As epigenetic alterations in CRC affect the biological and clinical behavior of the tumors, understanding these changes can lead to biomarkers for prognosis and treatment of CRC. The three aims proposed in this study will characterize genome-wide epigenetic alterations in CRC and correlate these epigenetic changes with protein expression and biological function. Aim 1 will determine the role of a novel methylated gene, RET, on cell signaling and colon cancer progression. RET expression in CRC cell lines that carry methylated RET will be induced using the DNMT inhibitor, DAC. Gene reconstitution studies will investigate the effects of RET methylation on apoptosis and cell proliferation, as well as on the activation state of select cell signaling pathways. Aim 2 will perform a comprehensive epigenomic characterization of colorectal cancer molecular subtypes with clinically distinct behaviors: Microsatellite Unstable (MSI), Microsatellite stable (MSS) and CpG Island Methylator Phenotype (CIMP) tumors. The molecular subgroup of primary CRC and cell lines will be determined, and their methylation status characterized using DNA methylation arrays. Aim 3 will parallel Aim 2 and determine the proteome of colorectal cancer molecular subtypes in cell lines and primary tumor using antibody arrays. Results of the proteome characterization will be compared to the methylome characterization in Aim 2 to determine which genes are silenced in CRC and what associated proteomic changes occur with the methylation changes. These studies will provide critical information for identifying and developing biomarkers for more effective treatment of people with CRC.